Microplate reader

The production of CK-MB was determined using the commercial ELISA kit (Merck & Co Inc, USA). Briefly, the collected supernatants and the prepared standards were added to a 96-well plate, and then incubated for 1.5 hours at 37°C. Subsequently, the medium was removed and conjugate reagents were added, followed by incubation for 1.5 hours at 37°C. After adding the TMB solution and incubating it for 15 minutes, the stop solution was added. Lastly, the absorbance at 450 nm was measured with a microplate reader (Mindray, Shenzhen, China).

An ELISA containing a recombinant 29 kDa T. cruzi calcium-binding protein as antigen was used to analyze serum samples taken from pediatric patients, as previously described [11 (link)]. The purified protein was resuspended in carbonate buffer pH 9.6 at a concentration of 5 μg/ml and dispensed to 96-well polystyrene plates at a final volume of 50 μl per well and fixed overnight at 4°C. Plates were blocked with phosphate-buffered saline (PBS) and 5% milk for 1 hour at room temperature. Dilution of the sera was performed in PBS and 1% milk at a concentration of 1/200. Incubation of sera was performed for 1 hour at 37°C. After 3 washes with PBS-Tween 0.05%, it was incubated with the second antibody: anti-human immunoglobulin G (IgG) coupled with peroxidase (Invitrogen Life Technologies, Frederick, MD, USA). After washing with PBS-Tween 0.05%, developing was performed with O-phenylenediamine dihydrochloride (OPD) 0.4 mg/mL in a citrate buffer pH 5 with 0.024% hydrogen peroxide (H₂O₂). Optical density (OD) at 490 nm was measured in a microplate reader (Mindray, Shenzhen, PR China).

After different treatment strategies, cells were treated with 0.25 mg/ml MTT (Sigma, Missouri, USA) at 37°C for 3 h, followed by removing the medium and adding the dimethyl sulfoxide to produce blue formazan. Then, the microplate reader (Mindray, Shenzhen, China) was used to measure the absorbance at 630 nm [17 (link)].

ELISA was used to measure the production of TNF-α and IL-18 in colon tissues. Briefly, testing samples and standards were implanted in 96-well plates, followed by being added with the conjugate reagents to be incubated for 1.5 h. After adding the TMB solution to each well, samples were incubated at 37°C for 15 min. Lastly, the stop solution was added to terminate the reaction, followed by measuring the absorbance at 450 nm using the microplate reader (Mindray, Shenzhen, China).

D-glucose, glibenclamide, and streptozotocin were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). A UV visible spectrophotometer (Gallenkamp PLC, UK) and microplate reader (Mindray, China) were used for spectrophotometric and enzyme linked immunosorbent assay (ELISA) measurements, respectively. Olympus CX 21 (Japan) microscope was used in the assessment of histopathology and immunohistochemistry of the pancreatic tissues.

To investigate the effect of HuR on the proliferation, migration and angiogenesis of HUVECs, cell counting kit-8 assay (CCK-8), scratch wound healing assay, and tube formation assay in Matrigel were performed. Briefly, the 3 groups of HUVECs were seeded at a density of 5 × 10³ cells per well with exosome-DMEM. The CCK-8 (Dojindo, Japan) was used to evaluate the cell proliferation. The optical density (OD) was measured at 450 nm by using a microplate reader (Mindray, China).
Scratch wounds were created by using 200 μL pipette tip at a cell confluency of 90%. The medium was replaced by exosome-DMEM. Photos were taken at 0, 12 and 24 hours afterwards. The residual area of the scratch wound was measured by using Image-Pro Plus 6.0 software (Media Cybernetics, USA).
Matrigel (BD Biosciences, USA) was used to assess the tube formation of HUVECs. The 3 groups of HUVECs were seeded at a density of 2 × 10⁴ cells per well into 24-well plates coated by Matrigel, and cultured in exosome-DMEM for 6 and 12 hours. Tube formation was assessed by phase-contrast microscopy (OLYMPUS, Japan) and the total tube length was measured using Image-Pro Plus 6.0 software.